This invention relates to analytical test devices and methods useful for analytical assays to determine the presence of analytes in fluid samples. It is especially useful for determining, the presence of cardiac analytes in whole blood, although it is not so limited.
The product and procedures of this invention can be utilized for many diagnostic purposes as well as for following the course of mammalian diseases and therapeutic treatments. It is applicable to many mammalian body fluids such as whole blood, serum, plasma and urine. Although this invention will be principally discussed as applied to detecting cardiac analytes it may also be applicable to other fields where antigen/antibody or equivalent reactions are utilized.
Many related assay procedures especially those including immunoassays may be performed using the device of the present invention and its disclosed modifications. For example, immunoassays or non-immunoassay test formats employing separation of red blood cells from plasma and a lateral fluid path may be employed. Analytes such as hormones for determining pregnancy or ovulation; viral, bacterial and fungal infectious microorganisms including H pylori for gastrointestinal ulcers, drugs of use and abuse and tumor markers are non-limiting examples. Enzymatic assays such as those which determine levels of glucose and other analytes in blood by formation of a chromogen are also contemplated by the present invention.
A number of immunoassay procedures have recently been developed which utilize reactions taking place on dry porous carriers such as cellular membranes through which samples to be analyzed can flow by capillary action, the reaction products being detectable either visually or with an instrument such as a reflectometer. While not so limited, these procedures generally involve antigen/antibody reactions in which one member of the reactive pair is labelled with a detectable label. Typically, the label is an enzyme label or a particulate direct label, for instance a sol label such as gold. The art is well aware of many useful labels and their method of operation.
Typical immunochromatographic devices of this nature are described in several United States and foreign patents. For example, U.S. Pat. No. 4,861,711 describes a device in which an analyte is detected by antigen/antibody reactions takings place in a series of coplanar membranes in edge to edge contact. Other devices are described in U.S. Pat. Nos.: 4,774,192; 4,753,776; 4,933,092, 4,987,065, 5,075,078, 5,120,643; 5,079,142; 5,096,809, 5,110,724, 5,144,890; 5,591,645; 5,135,716. All of these patents describe laminated structures.
Devices including cellular porous membranes such as those described in the above identified patents are often difficult to manufacture because they are multi-layer and require several layers of porous materials and filtration strips to insure accurate results.
For detection of cardiac analytes in whole blood, it is necessary to remove red blood cells so that they will not interfere with visualizing or otherwise detecting the colored reaction products normally produced in such immunoassay reactions.
Immunoassay devices when employed to detect cardiac analytes in whole blood utilize labelled antibodies which react with these antigens to produce detectable products. One widely utilized method for such diagnostic or analytical procedures utilizing antigen/antibody reactions employs a labelled detector antibody which reacts with one epitope on the antigen to form a labelled antibody/antigen complex formed in a detection zone of a porous membrane strip. The complex moves along the membrane by capillary action until it contacts a fixed line containing a capture antibody with which it reacts at another epitope on the antigen to concentrate and form a detectable reaction product. Typically, the product is visibly detectable because it is colored. With some constructions, the color is apparent to the naked eye. In more sophisticated devices, the presence or concentration of the antigen may be determined by measuring the intensity of the produced color or other property of the product with a suitable instrument, for example an optical sensor. The method is utilized in several devices used to detect cardiac analytes in whole blood. In all of these devices, it is necessary to prevent red blood cells from entering the color development or capture area because they interfere with proper visualization of the colored reaction product because of the intense hue of the cells.
Much effort has been expended to prevent such interference. As a result, products of this nature heretofore proposed for analysis of whole blood include some means, such as a type of filter to remove the red blood cells and form a plasma, so that there is no interference with the visibility of the color which is produced.
U.S. Pat. No. 5,135,716 utilizes an agglutinating agent to assist in the separation of red blood cells. Other patents describe the use of paper or plastic filters.
The use of glass fiber fleece is described in U.S. Pat. No. 4,477,575 to filter the red blood cells. Glass fiber fleece, however, simply adds another layer to the device. The principal difficulties arise from the problems of accurately placing several layers of thin flexible strips in proper registry in a laminar structure while at the same time retaining the sample placement zones, reaction zones and other areas of the membrane strips in proper communication with each other. The problems are further complicated by the difficulties of placing the completed membrane in or on a proper platform which is often a hollow casing with separable upper and lower members including fixed pillars and slots to prevent the membrane from moving and to retain selected membrane areas in proper position relative to viewing windows and other openings in the casing.
As a general rule, diagnostic devices such as those discussed above are often described as having an application zone to which the sample to be analyzed is added. The sample flows by capillary action along a predetermined pathway in a substrate, usually a nitrocellulose membrane, to a detection zone. The detection zone carries a mobile, labelled antibody to the analyte sought. If the analyte is present, a labelled antibody/analyte complex is formed which reacts with a fixed, i.e., immobilized capture antibody in a capture zone, downstream of the detection zone, to form a detectable product, usually one which is colored and visible to the naked eye.
It sometimes happens that the labelled antibody/analyte complex forms quite readily but does not sufficiently combine with capture antibody to produce an easily detectable signal. This might happen if no sufficient amount of complex contacts capture antibodies or contacts them in a configuration which is not optimum for forming a detectable reaction product. Other possible problems are insufficient incubation time or low antibody affinity.
These difficulties may be avoided by taking advantage of the biotin/avidin or biotin/streptavidin reaction or analogous reactions well known to the skilled artisan. These reactions are often used to increase the sensitivity of the diagnostic procedure.
In one application of this process, two antibodies are removably deposited in the detection zone and streptavidin is immobilized in the capture zone. The detector antibody is labelled, preferably with a metal such as gold, and reacts with one epitope on the analyte. The other antibody which is labelled with biotin reacts with another epitope on the analyte. The antibody mixture may be considered as a reagent system for use in detecting the presence of the analyte. If analyte is present, a complex containing (,old labelled detector antibody/analyte/biotin labelled detector antibody will form in the detection zone. The complex will move through a cellular membrane by capillary action to the capture zone. When the complex reaches the immobilized streptavidin in the capture zone, the streptavidin binds to the biotin and concentrates the complex in a small area to form a detectable reaction product.
There are several known variations of this reaction. For example, the detection zone may contain a biotin labelled antibody together with streptavidin labelled with a colored label such as gold. The complex which forms and moves into the capture zone is an analyte/biotin labelled antibody/streptavidin gold-labelled complex which will move to the capture zone and concentrate in the capture zone by reaction with a capture antibody to form a detectable reaction product.
The above identified procedures have generally been described to involve reactions taking place on an elongated, rectangular, laminated devices with the sample application zone at one end associated with some type of filter layer. The sample, after filtration, contacts a mobile, labelled specific binding reagent in a detection zone to form a complex which moves along a cellular membrane to a distally placed specific binding reagent, i.e., the capture reagent which is immobilized in a line across the membrane. The complex reacts with the reagent and is concentrated along the reagent line to become visible.
Typically, the sample to be analyzed is placed in the application zone by the addition of several drops to the center of the zone or by dipping the application zone into a small volume of the sample.
There are a number of problems with these configurations, especially when the goal is high sensitivity and the result should be visible within only a few minutes
High sensitivity can be achieved, for instance, by a capture line in a capture zone having a small width, as compared to the width of the detection zone, so that the amount of labelled reaction product is captured within a small capture area and thereby give a more intense signal color.
Further, the sensitivity can be increased as more labelled volume moves across the capture line during the test procedure. The more labelled volume is needed, however, the greater the area of the detection zone must be.
If this area has the form of an elongated channel and is increased by simply increasing the length thereof, the consequence is a considerable increase in test time, because the velocity of the moving liquid front slows down exponentially with the total distance wetted.
Other shapes of this area (e.g. with a higher ratio of width to length) leading to a large width detection zone and a small width capture zone channel have the disadvantage of creating stagnation regions where there is little or no flow. In extreme cases significant amounts of the sample may never become involved in the reactions which form the detectable product.
This invention alleviates many of the problems aforesaid by providing a device which may be small enough to be hand held, although not necessarily so, and provides for rapid and efficient flow of the fluid to be analyzed. Although its most important present utility is for the analysis of whole blood to diagnose for the presence of cardiac analytes, it may be adapted to test for the presence of other components in a fluid such as a body fluid carrying an antigen which will form a complex with an antibody which may thereafter be detected, for example in a sandwich assay with another antibody. Cardiac analytes as are described in several of the above-mentioned patents may be employed in the emergency room to aid the physician in diagnosing the cause of chest pain and to determine if the pain arises from a cardiac event.
It is towards several improvement in the features of the invention described above that the present application is directed.
The solution to these problems as explained herein is that, according to a first aspect of the present invention, the sample is allowed to enter into the detection zone simultaneously from many different directions and the detection zone is designed in a way that the resulting flow from the different directions all points to the entrance of the capture zone channel and all distances from entering the detection zone to said entrance are essentially the same.
This invention alleviates many of the problems aforesaid by providing a device which may be small enough to be hand held, although not necessarily so, and provides for rapid and efficient flow of the fluid to be analyzed. Although its presently preferred utility is for the analysis of whole blood to diagnose for the presence of cardiac analytes, it may be adapted to test for the presence of other components in a fluid such as a body fluid carrying an antigen which will form a complex with an antibody which may thereafter be detected, for example in a sandwich assay with another antibody.
Rapid and efficient flow of the fluid to be analyzed can be achieved by configuring the porous channels so that there is little or no opportunity for stagnation and so that the fluid enters a detection zone from a sample circulation channel from a multitude of points. The detection zone is designed so that the resulting front of the fluid moves in the direction of the entrance end of the capture zone.
A particular advantageous aspect of the invention when employed to whole blood is the selection of a porous substrate which chromatographically separates red blood cells from plasma. The chromatographic separation is of particular importance compared to filtration of particulate material because actual filtration may clog the cells of the media and impede or even stop flow. Moreover, as discussed above, filtration normally requires additional layers. In chromatographic separation, however, the particulate material continues to flow although at a slower rate than the carrier fluid so that there is little or no impedance of flow. With other biological samples chromatographic separation may not be necessary.
Another important feature of the invention, as will be explained below, is that all flow stops at a preselected point because the entire volume of the sample pathway in the porous carrier is wetted, and particulate material such as red blood cells does not interfere with the detection of the detectable reaction product.
The device according to a first aspect of this invention effects several improvements of the earlier devices, i.e. it provides a solution to the problems as explained herein is that the sample is allowed to enter into the detection zone simultaneously from many different directions and the detection zone is designed in a way that the resulting flow from the different directions all point to the entrance of the capture zone channel and all distances from entering the detection zone to said entrance are essentially the same. Rapid and efficient flow of the fluid to be analyzed can be achieved by configuring the porous channels so that there is little or no opportunity for stagnation and so that the fluid enters a detection zone from a sample circulation channel from a multitude of points. The detection zone is designed so that the resulting front of the fluid moves in the direction of the entrance end of the capture zone.
Further improvement can be archived with the following device according to a second aspect of the invention. For example, it uses less of the porous membrane, can be made smaller so that less material is used in its construction, and is faster acting. One of its most important advantages, as will be apparent from the following explanation, is that even when a plurality of analytes are to be identified, the only change in structure required is the structure of the porous membrane, and not the supporting layers.
The improvements herein are directed to the configuration of the device and the interaction between the porous membrane, on which the separation of plasma from blood cells occurs, and the top and bottom layers which cooperate to hold the membrane in the correct position. Furthermore, the top and bottom layers provide channels to conduct the sample from the application hole to and through the membrane to the capture antibodies while carrying out chromatographic separation of plasma from red cells. The first improvement is a reconfiguration of the sample delivery channel such that the fluid is conducted from the application hole to the membrane through a channel comprising the top and bottom layers of the device. In contrast to the device according to a first aspect of the invention, no membrane is present in the sample delivery channel. The absence of membrane at this location is an improvement in that it reduces the amount of porous membrane required for the device, and avoid concerns regarding the need to eliminate the porosity of membrane located in the sample delivery channel or any concerns regarding contact of the fluid sample with a material other than that comprising the top and bottom pieces (layers) of the device. The resulting product is less costly in both materials and labor to manufacture. The extent of porous membrane required is confined to the detector zone and capture zone A configured interface between the portion of the device comprising the sample delivery channel, and that containing the membrane and the sample circulation channel, is provided to form a capillary conduit for the blood sample to be channeled to the membrane in accordance with the device, without causing errant distribution of the sample.
A further advantage of this invention is that the same top and bottom layer components of the device are used in the manufacture of a number of different analytical tests. Only the membrane needs to be tailored for the detection of the specific analyte or analytes to be measured. For example, the reagents deposited or bound the membrane and their locations, and the shape of the fluid pathways on the membrane, can be individualized for each assay. The top and bottom layers with the sample delivery channel and sample circulation channel are the same for every assay.
The sample delivery channel may also be configured to contain a predetermined volume of sample, and further, by means of an optional window or transparency, indicate to the user when the sample delivery channel is full and thus adequate sample has been applied A further improvement is a configuration of the sample delivery channel such that when the channel is full, the sample therein contained is delivered to the sample circulation channel and thereby initiates the immunoassay.
In a further embodiment, reagents such as the labeled detector antibodies may be provided within the channels of the device in the fluid path prior to the membrane, such that the reagents mix with the sample. The reagents may be provided as beads, microbeads, or lyophilized powder, by way of non-limiting examples in the aforementioned channels.
A principal feature of the devices of this invention is that the membrane does not extend the full length of the sample delivery channel. Another is that the sample delivery channel is designed so that a known predetermined volume of sample can be delivered to the operation section of the device.
It is therefore an object of this invention to provide an analytical test device as described above with a sample delivery channel formed in the lower surface of the top layer and with walls defined by the channel and the top surface of the bottom layer. Almost no membrane is present in the region of the sample delivery channel. In one embodiment, the sample delivery channel is configured with parallel sides, and is in operative communication with the sample circulation channel. In another embodiment of the invention, the sample delivery channel is configured to contain the volume of sample needed to carry out the analysis in the device. In this embodiment, the end of the sample delivery channel which is in operative communication with the sample circulation channel is shaped to provide a narrowing of the sample delivery channel where it meets the sample circulation channel. In this embodiment, when the sample delivery channel has filled with fluid up to the point where the fluid contacts the narrowed section, capillary action will channel the fluid from the sample delivery channel to the sample circulation channel, and then onto the membrane of the device. The sample then flows until the sample delivery channel drains of its predetermined volume, and the analysis is performed. As mentioned above, an optional observation window at the junction of the sample delivery channel and the sample circulation channel may be provided to) indicate to the operator that adequate sample has been added to the device to conduct the test, as when the sample delivery channel has filled completely with blood and the sample is channeled to the sample circulation channel, the observation window will indicate the presence of the sample.
As in the case of the device according to a first aspect of the invention, the device according to a second aspect of the invention alleviates the problems with the prior art devices because the sample is allowed to enter into the detection zone simultaneously from many different directions and the detection zone is designed in a way that the resulting flow from the different directions all point to the entrance of the capture zone channel and all distances from entering the detection zone to said entrance are essentially the same. Rapid and efficient flow of the fluid to be analyzed is achieved by configuring the porous substrate (membrane) so that there is little or no opportunity for stagnation and so that the fluid enters a detection zone from a sample circulation channel from a multitude of points. The detection zone or channel is designed so that the resulting fluid front moves in the direction of the entrance end of the capture zone channel.
Even further improvement can be archived with the following device according to a third aspect of the invention. The improvements of this device according to the third aspect of the invention are also directed to the configuration of the device and the interaction between the porous membrane, on which the separation of plasma from blood cells occurs, and the top and bottom layers which cooperate to hold the membrane in the correct position. The present application is directed to devices in which the sample delivery channel is located on the top surface of the top layer, covered by a covering, and the sample is conducted to the sample circulation channel through a channel extending from the end of the sample delivery channel in the top surface of the top layer to the sample circulation channel. When the sample enters and fills the sample circulation channel, it then moves chromatographically onto the membrane simultaneously from a plurality of points and initiates the chromatographic separation of plasma from red cells and the entry of the fluid into the detection zone from a multitude of points. The sample delivery channel of the present invention, by virtue of its location on the upper surface of the top layer, offers several advantages. One advantage is the reduction in amount of membrane required in the device. The absence of membrane at this location is an improvement in that it reduces the amount of porous membrane required for the device, and avoid concerns regarding the need to eliminate the porosity of membrane located in the sample delivery channel or any concerns regarding contact of the fluid sample with a material other than that comprising the top and bottom pieces (layers) of the device. The resulting product is less costly in both materials and labor to manufacture. A second advantage is that the location of the channel allows the user to view the filling, of the channel. The test will not begin until the channel is filled, thus, no external sample measuring device is required. If the volume of the sample delivery channel is equal to the amount of sample required to conduct the test, when the channel is filled, further application of sample may be stopped. Furthermore, the sample collection portion of the device may be shaped to conveniently access a drop of blood obtained by finger prick, filling the sample delivery with a small volume of blood, generally 30 to 50 xe2x96xa1l, and initiating the assay.
A further advantage of the device according to a third aspect of the present invention is that a reagent may be placed in the sample delivery channel, in the form of an applied layer or one or more solid particles, which will dissolve in the sample as it passes through the channel. Application of reagents at this location provides an easier means for manufacture of the device, as well as allowing the reagent to mix with the sample early before the sample reaches the membrane.
Still a further advantage of this device according to a third aspect of the invention is that the same top and bottom layer components of the device are used in the manufacture of a number of different analytical tests. Only the membrane needs to be tailored for the detection of a specific analyte of analytes to be measured. For example, the reagents deposited or bound the membrane and their locations, and the shape of the fluid pathways on the membrane, can be individualized for each assay. The top and bottom layers with the sample delivery channel and sample circulation channel are the same for every assay.
As mentioned above, the sample delivery channel may also be configured to contain a predetermined volume of sample, and indicate to the user when the sample delivery channel is full and thus adequate sample has been applied. A further improvement is a configuration of the sample delivery channel such that when the channel is full, the sample therein contained is delivered to the sample circulation channel and thereby initiates the immunoassay. An additional, optional feature is a test end indicator which indicates that the test is complete and may be read, and obviates the need for a timer. The windows allowing the use to view the test results and test end indicator may be openings in the top layer of the device, or the entire device may be constructed of a transparent material which is opaqued by printing or surface treatment at the areas not to be viewed.
A principal feature of the devices of this third aspect of the invention is the location of the sample delivery channel on the top surface of the top layer of the device, such that the membrane does not extend the full length of the sample delivery channel. Another feature is that the sample delivery channel is designed so that a known predetermined volume of sample can be delivered to the operation section of the device A further feature is the placement of reagents within the sample delivery channel for dissolution in the sample.
It is therefore an object of this invention to provide an analytical test device as described above with a sample delivery channel formed in the top surface of the top layer. The sample delivery channel is covered by a cover, preferably transparent. No membrane is present in the region of the sample delivery channel. In one embodiment, the sample deliver channel is configured with parallel sides, and is in operative communication with the sample circulation channel. In another embodiment of the invention, the sample delivery channel is configured to contain the volume of sample needed to carry out the analysis in the device. In this embodiment, the end of the sample delivery channel which is in operative communication with the sample circulation channel is shaped to provide a narrowing of the sample delivery channel where it meets the sample circulation channel. In this embodiment, when the sample delivery channel has filled with fluid up to the point where the fluid contacts the narrowed section, capillary action will channel the fluid from the sample delivery channel to the sample circulation channel, and then onto the membrane of the device. The sample then flows until the sample delivery channel drains of its predetermined volume, and the analysis is performed.
As described herein, the device according to a third aspect of the alleviates the problems with the prior art devices because the sample is allowed to enter into the detection zone simultaneously from many different directions and the detection zone is designed in a way that the resulting flow from the different directions all point to the entrance of the capture zone channel and all distances from entering the detection zone to said entrance are essentially the same. Rapid and efficient flow of the fluid to be analyzed is achieved by configuring the porous substrate (membrane) so that there is little or no opportunity for stagnation and so that the fluid enters a detection zone from a sample circulation channel from a multitude of points. The detection zone is designed so that the resulting fluid front moves in the direction of the entrance end of the capture zone channel.